PROJECT SUMMARY Heterochromatic domains, such as centromeres, telomeres and other satellite DNA, pose a major challenge for DNA replication. Compacted chromatin is thought to inhibit replication initiation and obstruct progression of the replication machinery. Not surprisingly, recent analyses of various somatic and cancer cells have revealed that repressive chromatin is associated with regions of late replication and high mutation density. Little, however, is known about the molecular mechanisms that control and facilitate DNA replication at heterochromatic domains. Of importance, cancer cells may harness these mechanisms to facilitate heterochromatin replication and sustain their increased proliferative demands. For example, overexpression of KDM4A/JMJD2, a demethylase that removes the heterochromatic mark H3K9 tri-methylation, enables chromatin de-compaction and accelerated replication in ovarian cancers. Strikingly, KDM4A overexpression also resulted in copy gain of specific genomic loci often amplified in ovarian cancers and multiple myeloma, further strengthening the link between de-regulated heterochromatin replication and genomic instability. These observations highlight the need to elucidate the mechanisms required for proper heterochromatin replication so as to understand fundamental aspects of genome maintenance and the control of cell proliferation. This proposal is centered on METTL13 (Methyltransferase-like 13), a member of a poorly understood family of proteins containing putative SAM (S-adenosylmethionine)-binding domains. While METTL13 was found amplified and overexpressed in cancers, virtually nothing was previously known about its cellular functions. Preliminary results presented here provide the first insights on how METTL13 sustains cell proliferation, revealing crucial roles in DNA replication and chromatin dynamics. More specifically, we find that METTL13 is a novel key mediator of heterochromatin replication that is particularly important for replication of centromeres. To the best of our knowledge this is the first described regulator of human centromere replication timing. This proposal will combine cutting edge genomic and proteomic techniques with biochemical and cell biological approaches to dissect the action of METTL13 and establish its role in controlling chromatin dynamics, DNA replication and genome integrity. Generated results will reveal a fundamental mechanism of heterochromatin replication and replication timing control, and will establish novel drug targets for modulating chromatin dynamics and the proliferative capacity of cancer cells.